PROJECT SUMMARY Approximately 1% of children are born with a congenital heart defect, with half requiring medical and/or surgical treatment. Children born with congenital heart defects that result in increased pulmonary blood flow (PBF) and pressure develop abnormal pulmonary vascular reactivity. Although survival for these children has improved they continue to suffer morbidity and late mortality. This is due to the fact that they are at great risk for developing pulmonary vascular disease. In fact, even early pulmonary endothelial dysfunction, with abnormal vascular reactivity, causes significant morbidity and mortality. From our ongoing studies, it has become clear that there is an intimate, and complex, relationship between cellular metabolism and endothelial NO synthase (eNOS) signaling. However, how these processes are linked is unresolved and unravelling novel connections will be the major goal of this Project. Our group is at the fore-front of investigating the role played by post-translational modifications (PTMs) in the metabolic reprogramming associated with pulmonary vascular disease. The overall hypothesis we will test in this project is that PTMs play a major role in the metabolic reprogramming and loss of NO signaling associated with increased PBF and pressure. Using an experimental lamb model of CHD with increased PBF (Shunt), we have found that the mitochondrial redistribution of eNOS leads to the disruption of mitochondrial bioenergetics. Further, the resulting decrease in ATP generation leads to the proteasomal degradation of GTP cyclohydrolase I (GCH1), the rate limiting enzyme in the generation of the important NOS co-factor, tetrahydrobiopterin (BH4), secondary to the loss of hsp90 activity. We have identified the relevant E3 ubiquitin ligase as the carboxy-terminus of Hsc70 interacting protein (CHIP). However, a significant knowledge gap exists in our understanding of how mechanical forces induce the mitochondrial redistribution of eNOS and the activation of CHIP. Specific Aim 1 will fill this important gap by investigating the role played by the PTM- mediated activation of Akt1 and identifying ubiquitin proteasome pathway (UPP) components that are stimulated by mechanical forces. The tyrosine kinase, pp60Src is also chaperoned by hsp90 and has been shown to regulate mitochondrial bioenergetics. However, its role in regulating endothelial cell metabolism is not known. Thus, Specific Aim 2 will investigate whether the attenuation of mitochondrial bioenergetics associated with increased PBF and pressure also involves a decrease in pp60Src activity. To begin the clinical translation of our basic investigations, Specific Aim 3 will utilize our Shunt lamb model to determine if preserving mitochondrial bioenergetics or inhibiting the proteasome are therapeutic targets for enhancing NO signaling and endothelial function in children born with congenital heart defects that increase PBF and pressure. The successful completion of our proposed studies should yield new mechanistic insights and identify new targets that are amenable to therapeutic intervention.